Monoclonal antibodies for human non-small cell lung carinomas

ABSTRACT

The present invention is concerned with novel monoclonal antibodies which define antigens associated with human non-small cell lung carcinomas (&#34;NSCLC&#34;). The antibodies bind to normal human cells to a lesser degree than to tumor cells. The antibodies find use in diagnostic methods such as the detection of malignant cells associated with NSCLC and in therapeutic methods.

This is a continuation of application Ser. No. 667,521, filed Nov. 2,1984, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Human lung carcinomas are responsible for most deaths from cancer amongmen and are in the process of overtaking breast carcinomas as the mostfrequent cause of cancer death among women (Cancer Facts and Figures,1983). This disease can be divided into 4 major histological types,i.e., epidermoid (30%), adenocarcinoma (35%), large-cellundifferentiated (15%), and small-cell (20%). Most cases of lungcarcinomas are incurable by chemotherapy and radiation therapy. Smallcell lung carcinomas may respond to chemotherapy and radiation therapyby a reduction in size, but not a total cure. Complete surgical removalof the tumor appears to be the only effective therapy. Unfortunately,however, fewer than 30% of lung cancer patients have tumors which can betotally resected at diagnosis and of these, fewer than one-third survive5 years after apparent complete surgical removal of all tumor. There isa great need, therefore, for methods that would make possible an earlierdiagnosis of lung cancer, a better definition of the degree of cancerspread, and a more effective therapy.

Monoclonal antibodies may be used for all these purposes. Aprerequisite, however, is to find antibodies to antigens that are morestrongly expressed in lung cancer than in normal adult tissues. In viewof the known heterogeneity of tumor cells populations, the presence ofseveral determinants on the same antigen molecule, the anticipateddifferences between antigens with respect to their suitability asdiagnostic markers as compared to therapeutic targets, and the differentbiological characteristics of different antibodies to the same antigen,a number of different antibodies may be needed.

2. Description of the Prior Art

Human monoclonal antibodies to lung cancer antigens are described bySikora et al., Br. J. Cancer (1981) 43: 696-700. Monoclonal antibodiesthat demonstrate specificity for several types of human lung cancer aredisclosed by Cuttitta et al., Proc. Natl. Acad. Sci. U.S.A. (1981) 78:4591-4595. Antigens associated with a human lung adenocarcinoma definedby monoclonal antibodies are described by Varki et al., Cancer Research(1984) 44: 681-687.

Continuous cultures of fused cells secreting antibody of predefinedspecificity are described by Kohler et al., Nature (1975) 265: 495-497.

SUMMARY OF THE INVENTION

The present invention is concerned with novel monoclonal antibodieswhich define determinant sites on antigens of human non-small cell lungcarcinoma (NSCLC) cells. The term "NSCLC cells" includes epidermoidcarcinoma cells, adenocarcinoma cells, and large cell undifferentiatedcarcinoma cells. The determinant sites may also be found on antigens ofsome other carcinomas, e.g. some carcinomas of the breast, and, thus,the antibodies of the invention will also bind to these other carcinomacells. These monoclonal antibodies bind to a lesser degree to normaladult cells than to tumor cells. The term "bind to a lesser degree"means that the binding will not be detectable by immunohistologicaltechniques or that the binding will be about four to five times lessthan the binding to NSCLC cells as determined by immunohistologicaltechniques. The monoclonal antibodies are secreted by murine hybridomas.

The invention also concerns certain diagnostic methods employing themonoclonal antibodies of the invention. One such method involves thedetermination of the presence of NSCLC cells in a specimen suspected ofcontaining such cells. The specimen is contacted with the monoclonalantibody, which is capable of distinguishing such cells from other celltypes which may be present in the specimen. The contact is carried outunder conditions for binding of the antibody to such cells. Aftercontact, the presence or absence of binding of the antibody to the cellsin the specimen is determined. This binding is related to the presenceor absence of the NSCLC cells in the specimen. Generally, the specimenis contacted with a labeled specific binding partner for the monoclonalantibody. This label is capable of producing a detectable signal.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention concerns certain novel antibodies specific forantigens on human NSCLC cells and certain diagnostic methods employingsuch antibodies. The monoclonal antibodies of the invention may beproduced according to the standard techniques of Kohler and Milstein,supra. For example, human lung carcinoma cells from plural effusions orcultured cells from human non-small cell lung carcinoma, or cells from anormal fetal lung, are used as the immunogen. These cells are injectedinto a mouse and, after a sufficient time, the mouse is sacrificed andspleen cells obtained. The spleen cell chromosomes encoding desiredimmunoglobulins are immortalized by fusing the spleen cells with myelomacells or with lymphoma cells, generally in the presence of polyethyleneglycol. The resulting cells, which include the fused hybridomas, areallowed to grow in a selective medium, such as HAT-medium, and thesurviving cells are grown in such medium using limiting dilutionconditions. The cells are grown in a suitable container, e.g.,microtiter wells, and the supernatant is screened for monoclonalantibodies having the desired specificity.

Various techniques exist for enhancing yields of monoclonal antibodies,such as injection of the hybridoma cells into the peritoneal cavity of amammalian host, which accepts the cells, and harvesting the ascitesfluid. Where an insufficient amount of the monoclonal antibody collectsin the ascites fluid, the antibody is harvested from the blood of thehost. Various conventional ways exist for isolation and purification ofthe monoclonal antibodies, so as to free the monoclonal antibodies fromother proteins and other contaminants (see Kohler and Milstein, supra).

One such monoclonal antibody of the present invention is exemplified bya novel antibody designated L3. This monoclonal antibody defines adeterminant site on a cell associated protein antigen characteristic ofhuman NSCLC cells, i.e. malignant cells, and a determinant site on aprotein antigen that is secreted into the culture medium by the NSCLCcells. When lysates of biosynthetically labeled target cells areimmunoprecipitated with L3 antibody and the precitates are submitted tosodium dodecyl sulfate-polyacrylamide one-dimensional gelelectrophoresis (SDS-PAGE), the cell associated protein has a molecularweight of about 80,000 daltons. By the same method the secreted proteinhas a molecular weight of about 100,000 daltons. The antibody is of theIgG₁ isotype. The L3 antibody binds to a lesser degree to some normalcells. The L3 antibody is produced by the L3 murine hybridoma.

Another monoclonal antibody of the invention, designated L5, defines adeterminant site on a cell surface protein antigen characteristic ofhuman NSCLC cells. The molecular weight of the protein, as determined bythe method described above and by a method based on ¹²⁵ I labelling isabout 140,000 daltons. This antibody is of the IgM class. The L5antibody does not bind detectably to normal cells, such as fibroblasts,endothelial cells, and epithelial cells in the major organs. The L5antibody is produced by the L5 murine hybridoma.

Another monoclonal antibody of the invention is designated L18 anddefines a determinant site on another cell surface protein antigencharacteristic of human NSCLC cells. The molecular weight of theprotein, as determined by both of the methods described above, is about72,000 daltons. This antibody is of the IgG₁ isotype. The L18 antibodybinds to a lesser degree to some normal cells, particularly in thespleen. The L18 antibody is produced by the L18 murine hybridoma.

Also included within the scope of the invention are useful bindingfragments of the monoclonal antibodies above such as Fab, F(ab')₂, Fvfragments and so forth. The antibody fragments are obtained byconventional techniques. For example, useful binding fragments may beprepared by peptidase digestion of the antibody using papain or pepsin.

While the above specific examples of the novel antibodies of theinvention are directed to antibodies binding to specific determinantsites on the respective antigens and being of specific classes areisotypes from a murine source, this is not meant to be a limitation. Theabove antibodies and those antibodies having functional equivalency withthe above antibodies, whether from a murine source, mammalian sourceincluding human, or other sources, or combinations thereof are includedwithin the scope of this invention, as well as other classes such asIgM, IgA, IgE, and the like, including isotypes within such classes. Bythe term "functional equivalency" is meant that the antibody is capableof binding to the above-described determinant site and capable ofcompeting with a particular antibody of the invention for such site.That is, such antibody, when combined with a specimen containing a cellor cell fragment having such determinant site, will bind to suchdeterminant site and will block an antibody of the invention frombinding to such site. Furthermore, since the various antigens of theinvention can have more than one determinant site, the inventionincludes monoclonal antibodies which define determinant sites on thoseantigens other than determinant sites defined by the aforementionedmonoclonal antibodies.

The invention also includes the aforementioned antigens in purifiedform. The antigens can be purified by conventional methods such asimmunoprecipitation as described in Brown et al., Proc. Natl Acad. Sci.U.S.A. (1981a) 78: 539-543.

One method of the invention involves the determination of the presenceof a malignant condition in lung tissue. The term "malignant condition"refers to the presence of dysplastic including carcinoma in situ,neoplastic, malignant, or tumor cells, or the like. The specimen iscontacted or combined with a monoclonal antibody of the invention. Thecontact is carried out under conditions for binding of the antibody tothe malignant cells. After contact, the presence of binding of theantibody to the malignant cells in the specimen is observed. That is,the specimen is examined for immune complexes of the antibody and theantigenic site. This immune complex formation is related to the presenceof malignant cells in the specimen.

A particular example, by way of illustration and not limitation, of amethod in accordance with the invention is a method for the detection oftumor cells in excised tissue. The above method is applied to a specimenwhich is a section of the tumor obtained after removal of the tumor. Thetumor that is excised is treated to obtain sections, which treatmentinitially involves freezing the tumor or tissue, normally freezingimmediately after excision. The frozen layer of tissue is then cut intosections using, for example, a cryostat.

The section of the tumor obtained as described above is contacted with amonoclonal antibody of the invention and then with a second antibodydirected against the above monoclonal antibody, which second antibody islabeled with a detectable label.

The excised specimen, e.g., the section of the tumor, is contacted withthe first monoclonal antibody under conditions for binding of theantibody to the malignant cells. The incubation is generally conductedin an aqueous medium such as, for example, phosphate buffered salinecontaining a small amount of sodium azide, in a suitable container suchas, for example, a glass petri dish, for a period from about 15 to 30minutes at a temperature of from about 20° to 30° C. The amount ofantibody employed is usually sufficient to provide detectable binding,i.e., to provide a detectable number of immune complexes between theantibody and the determinant or antigenic site in question.

Following the incubation, the section is washed to reduce or eliminatenon-specifically bound antibody and then is examined to observe theabove-mentioned complexes which result from binding of the monoclonalantibody to the cells of the specimen possessing the antigenic site. Thebinding is related to the presence of malignant cells in the section.Accordingly, binding is determined, for example, by contacting thespecimen with a labeled specific binding partner for the monoclonalantibody. The label is capable of producing a detectable signal and maybe a radioactive label, a chromophore such as a fluorescer, an enzyme,or the like.

An example of a technique employing the above approach isimmunofluorescence staining. In this technique frozen sections of thetumor are fixed on a glass slide with acetone and are incubated with themonoclonal antibody in, for example, a petri dish. After washing with anappropriate buffer such as, for example, phosphate-buffered saline, thesection is placed on a petri dish and contacted with the labeledspecific binding partner for the monoclonal antibody, which may be, forexample, a labeled antibody specific for the monoclonal antibodyemployed. Since, for the most part, the monoclonal antibody will bederived from a murine source, a labeled antimouse immunoglobulinspecific for the monoclonal antibody may be employed. Suchimmunoglobulins may be raised according to standard techniques byinjecting a suitable host with murine antibody, waiting for anappropriate time, and harvesting the antimouse immunoglobulins from theblood of the injected host.

After a second washing of the slide with, for example, an aqueousbuffer, the sections may be covered with a fluorescent antibody mountingfluid and a coverslip and then examined with a fluorescence microscopeto determine the binding of the monoclonal antibody to the section. Thedetermination of the binding also may include an identification of thelocation of such binding within the specimen.

The binding of the monoclonal antibody to the specimen may also bedetermined by employing a monoclonal antibody which is covalentlyconjugated to a label capable of producing a detectable signal, such asa radioactive entity, a chromophore including dyes and fluorescers, oran enzyme. The number of labels employed per antibody is generallydetermined by the requirements of the diagnostic method in which thelabeled antibody is employed and the availability of sites for linkingthe label to the antibody.

Methods for conjugating labels to antibodies and antibody fragments arewell-known in the art. Such methods may be found in U.S. Pat. Nos.4,220,450; 4,235,869; 3,935,074; and 3,996,345.

Another example of a technique in which the monoclonal antibody of theinvention may be employed is immunoperoxidase labeling (Sternberger,Immunocytochemistry, John Wiley & Sons, New York, 1979, pp: 104-169 asmodified by Garrigues et al., Int. J. Cancer (1982) 29: 511-515). Thetissue to be tested is fixed with a suitable solvent, such as acetone,on a support, such as a glass slide. Next, the tissue is incubated withthe monoclonal antibody and then washed free of unbound antibody. Then,the tissue is incubated with rabbit anti-mouse IgG, washed to removeunbound antibody, combined with mouse peroxidase-anti-peroxidasecomplex, washed to remove unbound conjugate, and then treated withsubstrate for the enzyme. Following this treatment the slide is examinedfor a detectable signal.

The antibodies of the invention may be used in a method of determiningthe presence of a malignant condition in an exfoliative cell specimenfrom the lung, such as sputum. By the term "exfoliative" is meant thatthe specimen comprises isolated cells or clumps of cells obtained byscraping or washing the surface of tissue, which cells are removedindividually or in scales or laminae. The exfoliative cell specimen isto be distinguished from excised tissue such as that obtained by biopsy.Contact between the specimen and the antibody is made under conditionsfor binding of the antibody to the antigenic site. After contact, thepresence or absence of binding of the antibody to the antigenic site isdetermined and is related to the presence of a malignant condition inthe lung.

To determine the presence of a malignancy in the lung, a sputum samplewould provide the exfoliative cell specimen to be used in the method.The method may find utility in the detection of a malignant condition inexfoliative cell specimens from the bronchus, gastro-intestinal tractincluding oral pharynx, mouth, etc.

The exfoliative cell specimen is next contacted with the aforementionedantibody under conditions for binding of the antibody to the specificantigenic site in the specimen to form antigen-antibody complexes. Thisantigenic site may be present on cells or cell fragments in thespecimen. Generally, the specimen is placed on an appropriate support,such as, for example, a slide, usually glass, or some other suitablematerial. The exfoliative cell specimen is generally smeared on theslide to provide a thin layer of the specimen on the surface of theslide. The contact between the antibody and the specimen is generallycarried out in an aqueous buffered medium. The buffers which may beemployed include phosphate, tris, bicarbonate, etc. The pH is related tothe nature of the specimen and the antibody, and is generally in therange of from about 5 to 8. The aqueous medium may additionally containorganic polar solvents in an amount of from about 0 to 40%. The organicpolar solvents are water soluble and generally have from about 1 to 10carbon atoms and from about 1 to 4 oxygen atoms. The antibody will bepresent in the aqueous medium at a concentration of about 1 to 100μg/ml, preferably from about 10 to 20 μg/ml. The temperature during thecontact of the specimen with the antibody is usually from about 4° to40° C., preferably about 10° to 30° C. The period of contact is usuallyfrom about 15 to 120 minutes, preferably from about 30 to 60 minutes.

After the period of contact between the specimen and the antibody, thesupport is generally treated to remove unreacted antibody. Normally,this is accomplished by washing the support with an aqueous, usuallybuffered, medium. In general, the amount of wash solution should besufficient to remove the unreacted antibody.

Next, the presence of binding of the antibody to the antigenic site inthe specimen, which binding is related to the presence of a malignantcondition at the locus, is observed. That is, the specimen is examinedto determine the number of antigen-antibody (immune) complexes formed.It should be noted that in some instances very small numbers of theantigenic site in question may be found in the exfoliative cellspecimen. However, in a malignant condition, large numbers of theantigenic site will be present and this latter condition is readilydistinguishable by this method over a non-malignant condition because alarge number of antigen-antibody complexes will be measurable where amalignant condition exists. To make the determination of the presence ofbinding, means for producing a detectable signal is incorporated intothe assay system. For example, one may conjugate the antibody employedin the assay to a label which is capable of producing a detectablesignal. The label may be a radioactive entity, a chromophore includingdyes and fluorescers, an enzyme, or the like. The number of labelsemployed for the antibody is generally determined by the requirements ofthe method and the availability of sites for linking the label to theantibody.

Alternatively, one may contact the washed slide with a labeled specificbinding partner for the antibody, which may be, for example, a labeledantibody specific for the antibody employed. Where the monoclonalantibody is derived from a murine source, a labeled anti-mouseimmunoglobulin specific for the antibody employed in the method may beused. Such immunoglobulins may be raised according to standardtechniques by injecting a suitable host with the monoclonal antibody,waiting for an appropriate time, and harvesting the anti-mouseimmunoglobulins from the blood of the injected host. When a labeledspecific binding partner for the antibody is employed, the slide must bewashed again with an aqueous medium prior to examining the slide forfluorescence.

To determine the presence of binding between the antibody and the cellspecimen where a fluorescer label is used, one may examine the slide forfluorescence, usually employing a fluorescence microscope. Where a labelother than a fluorescer is employed, one may examine the slide or thespecimen for the formation of a precipitate, a color, or the like.

The above description is directed primarily to the use of the antibodiesof the invention in immunofluorescence techniques. However, theantibodies of the invention may be used in most assays involvingantigen-antibody reactions. The assays may be homogeneous orheterogeneous. In a homogeneous assay approach, the specimen may bebiological fluid such as serum, urine, and the like or the specimen maybe lysed and clarified to remove debris. Antibody L3, for example, hasbeen used by us to measure the concentration of its cognate antigen inserum from cancer patients, and certain cancer patients have been foundto have elevated levels of this antigen when compared to normalcontrols. The immunological reaction usually involves the specificantibody, a labeled analyte, and the sample of interest. The signalarising from the label is modified, directly or indirectly, upon thebinding of the antibody to the labeled analyte. Both the immunologicalreaction and detection of the extent thereof are carried out in ahomogeneous solution. Immunochemical labels which may be employedinclude free radicals, fluorescent dyes, enzymes, bacteriophages,coenzymes, and so forth.

In a heterogeneous assay approach, the reagents are usually thespecimen, the specific antibody, and means for producing a detectablesignal. The specimen is generally placed on a support, such as a plateor a slide, and contacted with the antibody in a liquid phase. Thesupport is then separated from the liquid phase and either the supportphase or the liquid phase is examined for a detectable signal employingmeans for producing such signal. The signal is related to the presenceof the analyte in the specimen. Means for producing a detectable signalincludes the use of radioactive labels, fluorescers, enzymes, and soforth. Exemplary of heterogeneous immunoassays are the radioimmunoassay,immunofluorescence methods, enzyme-linked immunoassays, and the like.

For a more detailed discussion of the above immunoassay techniques, see"Enzyme-Immunoassay," by Edward T. Maggio, CRC Press, Inc., Boca Raton,Fla., 1980. See also, for example, U.S. Pat. Nos. 3,690,834; 3,791,932;3,817,837; 3,850,578; 3,853,987; 3,867,517; 3,901,654; 3,935,074;3,984,533; 3,996,345; and 4,098,876, which listing is not intended to beexhaustive.

The antibodies of the invention can also be employed to image metasticdeposits in human patients with NSCLC in a manner analogous to thatdescribed for malignant melanoma in J. Nucl. Med. (1983) 24: 123-129 andin J. Clin. Invest. (1983) 72: 2101-2114. The antibody or fragmentsthereof are radiolabelled and administered intraveneously to a patientwho subsequently is imaged using, e.g., a gamma camera or the like.

The invention also includes diagnostic kits for carrying out the methodsdisclosed above. In one embodiment, the diagnostic kit comprises (a) amonoclonal antibody more specifically defined above and (b) a conjugateof a specific binding partner for the above monoclonal antibody and alabel capable of producing a detectable signal. The reagents may alsoinclude ancillary agents such as buffering agents and proteinstabilizing agents, e.g., polysaccharides and the like. The diagnostickit may further include, where necessary, other members of the signalproducing system of which system the label is a member, agents forreducing background interference in a test, control reagents, apparatusfor conducting a test, and the like. In another embodiment, thediagnostic kit comprises a conjugate of a monoclonal antibody of theinvention and a label capable of producing a detectable signal.Ancillary agents as mentioned above may also be present.

The antibodies of the invention may be used therapeutically. Antibodieswith the proper biological properties are useful directly as therapeuticagents. Alternatively, the antibodies can be bound to a toxin to form animmunotoxin or to a radioactive material or drug to form aradiopharmaceutical or pharmaceutical. Methods for producingimmunotoxins and radiopharmaceuticals of antibodies are well-known (see,for example, Cancer Treatment Reports (1984) 68: 317-328).

Another therapeutic use of the monoclonal antibodies of the presentinvention is the immunization of a patient with an anti-idiotypicantibody raised by using one of the present monoclonal antibodies as animmunogen. Such immunization can induce an active anti-tumor activity(see, for example, Nepom et al.; Proc. Natl. Acad. Sci. U.S.A. (1984)81: 2864-2867. In a similar approach, the patient can be immunized withthe antigen in purified form, a peptide fragment of the antigen, or amodified form of the antigen.

A particularly attractive aspect of the present invention is that acombination of two or more of the present antibodies can be employed.The combination is effective in detecting at least the four types oflung carcinomas mentioned above, namely, large cell undifferentiatedlung carcinoma, small cell lung carcinoma, adenocarcinoma, andepidermoid carcinoma. For example, monoclonal antibodies L3 and L18 canbe combined in effective amounts, i.e., amounts that will render thecombination capable of detecting all of the aforementioned lungcarcinomas.

Some of the monoclonal antibodies of the invention also definedeterminant sites on antigens associated with other carcinomas such asbreast carcinoma and epidermoid carcinomas of the vulva. Consequently,the present antibodies can find use in diagnostic and therapeuticproducts directed to such carcinomas.

EXAMPLES

The invention is further demonstrated by the following illustrativeExamples. A number of procedures employed will be described first.

Immunohistological Technique

For immunohistological studies on frozen sections, the unlabelledantibody technique of Sternberger in Immunochemistry, John Wiley & Sons,New York, 1979, pp: 104-169, as modified by Garrigues et al. in Int. J.Cancer (1982) 29: 511-515, was used. The target tissues for these testswere obtained at surgery and frozen within 4 hr of removal in isopentanewhich had been precooled in liquid nitrogen. Tissues were then stored inliquid nitrogen or at -70° C. until use. Rabbit anti-mouse IgG(Sternberger-Weyer Immunochemicals, Inc., Jarettsville, MD) was used ata dilution of 1/50. Mouse peroxidase-antiperoxidase complex (PAP,Sternberger-Meyer Immunochemicals, Inc.) containing 2 mg/ml ofspecifically purified PAP was used at a dilution of 1/80. Frozensections were prepared, dried, treated with acetone and dried (Garrigueset al., 1982). Sections to be used for histologic evaluation werestained with hematoxylin. To decrease nonspecific background, sectionswere preincubated with normal human serum diluted 1/5 (Garrigues et al.,1982). Mouse antibodies, goat anti-mouse IgG, and mouse PAP were dilutedin a solution of 10% normal human serum and 3% rabbit serum.

The staining procedure consisted of treating serial sections with eitherspecific or control antibody for 2.5 hr, incubating for 30 min withrabbit anti-mouse IgG diluted 1/50, and exposing for 30 min to mouse PAPcomplex diluted 1/80. After each treatment with antibody, the slideswere washed twice in phosphate buffered saline (PBS). Theimmunohistochemical reaction was developed with freshly prepared 0.05%3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) and0.01% hydrogen peroxide in 0.05M Tris buffer, pH 7.6 for 8 min. Furtherexposure to a 1% OsO₄ solution in distilled water for 20 min intensifiedthe stain. The sections were rinsed with water, dehydrated in alcohol,cleared in xylene, and mounted on slides.

The slides were each read under code and coded samples were checked byan independent investigator. Typical slides were photographed by usingdifferential interference contrast optics (Zeiss-Momarski). The degreeof antibody staining was evaluated as 0 (no reactivity), + (few positivecells), ++ (at least one third of the cells positive), +++ (most cellspositive), ++++ (close to all cells strongly positive). Sincedifferences between + and 0 staining were less clear cut than between ++and + staining, it was decided to count as "positive" a staining gradedas ++ or greater. Both neoplastic and stroma cells were observed intumor samples; the staining recorded referred to that of the tumorcells, since the stroma cells were not stained at all, or were stainedmore weakly than the tumor cells.

Determination of Antigen Location

The subcellular localization of antigens was determined by measuringantibody binding to cells before or after permeabilization withnon-ionic detergent. Antibodies binding to the cell surface of intactcultured cells were identified by either direct binding assays with ¹²⁵I-labelled antibody (Brown et al., Proc. Natl. Acad. Sci. U.S.A. (1981a)78: 539-543) or by indirect fluorescence using the (FACS) II cellsorter. Antibodies binding to intracellular locations were determined bydirect binding of ¹²⁵ I-labelled antibody to cells following fixationwith paraformaldehyde and subsequent permeabilization with the non-ionicdetergent NP-40.

Binding Assays

(a) For binding assays performed by using radiolabelled antibodies(Brown et al., supra), cultured cells (10⁶) were incubated at 4° C. for30 min with 10⁶ cpm of ¹²⁵ I-labelled antibody in 100 μl ofheat-activated (30 min at 56° C.) fetal calf serum in culture medium.After the addition of 5 ml of PBS, the cells were pelleted bycentrifugation for 10 min at 250×g. The supernatant was aspirated, andthe pellet was counted for ¹²⁵ I. To measure nonspecific binding,parallel incubations were performed with 10 μg of unlabelled antibody asa competitor (Brown, et al., supra). In some instances binding assayswere carried out in an analogous fashion on cells monolayers attached toplastic culture dishes.

(b) For binding assays performed on the FACS II cell sorter, cells wereremoved from their substrata using PBS containing a 5 mM ethylenediaminetetraacetic acid (EDTA). Samples containing 1×10⁵ cells were incubatedfirst with monoclonal antibody at a concentration of 2 μg/ml followed byfluorescein-conjugated goat anti-mouse antibody at a 1:200 dilution.Cells were then washed and resuspended in culture medium. Immediatelyprior to FACS analysis, propidium iodide was added to a finalconcentration of 1 μg/ml to stain non-viable cells. During FACSanalysis, cells emitting red fluorescence were electronically gated outso that only viable cells were examined. The mean intensity offluorescein fluorescence was then determined for each antibody. Negativecontrols consisted of samples in which monoclonal antibody was omitted;positive controls consisted of monoclonal antibodies to HLA type 1histocompatibility antigens. Staining was regarded as positive if themean channel fluorescein was at least 3 times background.

Protein Antigen Determination

In order to identify protein antigens, lung carcinoma or human embryoniclung cells were surface radioiodinated or metabolically labelled with ³⁵S-methionine. Antigens were isolated from cell lysates by incubationwith monoclonal antibody, addition of goat anti-mouse IgG, andadsorption to S. aureus. Immune precipitates were washed and analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)(10-20% acrylamide) as described (Brown et al., supra).

³⁵ S-Methionine Labeling of VM-2 Antibody

Hybridoma cells producing a particular antibody were seeded into amicrotiter well in methionine free Dulbecco's Minimum Essential Medium(DMEM) containing 25 mM Hepes buffer, 4 mM L-glutamine, 4.5 gm/lglucose, 10 mM non-essential amino acids, 100 units/ml penicillin, 100μg/ml streptomycin and 15% heat inactivated newborn calf serum (NCS).The cells were contacted for 6 hrs with 0.1 mCi ³⁵ S methionine at 37°C., the supernatant removed and centrifuged to remove cells.

Isotype Determination

(a) Ouchterlony immunodiffusion

An aliquot of supernatant of particular hybridoma cells was placed intothe center well of a 2% agar plate. Monospecific rabbit anti-mouse Igisotypes antibodies (Meloy) were placed in the outer wells and the platewas incubated for 2 h at room temperature and overnight at 4° C.

(b) Flexible polyvinylchloride 96 well plates (Costar) were coated with0.1 mg/ml goat anti-mouse Ig antibodies for 2 h at 37° C. andcountercoated with a 3% BSA solution for 2 h at 37° C. The hybridomasupernatant was then incubated at 37° C. for 2 h. After washing with PBSbovine serum albumin (BSA) plates were incubated at 37° C. for 2 h withmonospecific rabbit anti-mouse Ig isotype antibodies coupled toperoxidase (Zymed). After washing, plates were incubated with 1 mg/mlorthophenylenediamine and 0.03% H₂ O₂ in 0.1M citrate buffer pH 4.5.Optical density at 630 nm was determined on a Dynatec ELISA platereader.

Staphylococcal Protein A Binding Assay

Microtiter wells were incubated with 5% NCS in PBS plus 0.02% NaN₃ andthe supernatant was aspirated. Twenty-five μl of a suspension ofepidermal cells (2×10⁷ cells/ml) were added to each well and incubatedwith 25 μl of a particular antibody for 1 hr at room temperature. Theplates were centrifuged at 1200 rpm for 7 min, washed twice with 50%NCS/PBS/NaN₃ and 25 μl ¹²⁵ I-staphylococcal protein A (about 50,000cpm/25 l) were added. The plates were incubated for 1 hr at 25° C.,washed twice with 5% NCS/PBS/NaN₃ and dried. The bottom of the wellswere cut off and counted in a gamma counter.

EXAMPLE Preparation of Monoclonal Antibodies

Monoclonal antibodies were produced by immunizing 3-month-old BALB/cmice with human tissues of one of four different sources: (1) pleuraleffusions from patients with metastic non-small cell lung carcinoma, (2)cultured cells from a non-small cell lung carcinoma, and (3) lung tissuefrom 3-4 months-old human embryos. The immunizations were performed byinjecting the mice intraperitoneally 3-4 times with approximately 10⁷cells. Three days after the last immunization, the spleens were removed,suspended in culture medium and fused with NS1 mouse myeloma cells(Kohler and Milstein, supra). The mixtures were seeded to form lowdensity cultures originating from single fused cells (clones); thetechniques used for the hybridization have been previously described byYeh, et al., Int. J. Cancer (1979) 29: 269-275.

Supernatants from hybrid cells were screened by using both an ELISAassay and an autoradiographic indirect ¹²⁵ I-labelled protein A assay(Brown et al., J. Immunol. Meth. (1979) 31: 201-209 against extractsfrom the tumors used for immunization which contained, i.a., cellmembranes. These extracts were prepared using a procedure modified fromColcher et al., Cancer Res., (1981) 42: 1451-1459; Yeh et al., supra.For this, tissues were washed with PBS and suspended, which for intacttumors was done by pressing through a stainless steel screen. After this1 mM NaHCO₃ containing 1 mM phenylmethylsulfonylfluoride(Calbiochem-Behring Corp., San Diego, CA) was added, and the materialwas then homogenized on ice, with 50 strokes of the B pestle of a Douncehomogenizer. After centrifugation for 15 min at 27,000×g, thesupernatant was removed, and the pellet was resuspended in PBS,sonicated for 1 min, and stored at -70° C.

Hybridomas which produced antibodies binding to the cell membraneextracts were cloned, expanded in vitro, and further tested for antibodyspecificity. This testing was carried out by using theImmunohistological Technique described above, in which the ability ofthe antibodies to bind to frozen sections of lung carcinomas, othertumors and normal human tissues were tested. Those hybridomas whichproduced antibody of apparent specificity for human lung cancer wererecloned, expanded and injected into pristane-primed 3-month old BALB/cmice, where they grew as ascites tumors.

Antibodies secreted into the ascites were purified on protein ASepharose (Ey et al., Immunochemistry (1978) 15: 429) or by gelfiltration in Sephacryl S-300. Purified antibodies were used for furthercharacterization which included additional specificity tests byimmunohistology, binding assays on intact cells to determine whichantibodies bound to the cell surface, and the radioimmunoprecipitationtests as described above.

Monoclonal antibodies L3, L5, and L18 were produced from thecorresponding hybridomas as described above. These antibodies exhibitedthe properties indicated above in this specification.

The cell lines, designated L3, L5, and L18 were deposited at theA.T.C.C. (American Type Culture Collection, 12301 Park Lawn Drive,Rockville, MD 20852 USA) on Oct. 4, 1984, and received accession numbersHB8626, HB8627, and HB8628, respectively. Cell line L3 was redepositedon Oct. 25, 1984.

The invention has been described in detail with particular reference tothe above embodiments. It will be understood, however, that variationsand modifications can be effected within the spirit and scope of theinvention.

What is claimed is:
 1. A monoclonal antibody, the antigen-combining siteof which binds to:(a) a protein antigen having a molecular weight ofabout 80,000 daltons and which is a cell-surface determinant of humannon-small cell lung carcinoma; and (b) a protein antigen having amolecular weight of about 100,000 daltons and which is secreted by humannon-small cell lung carcinoma.
 2. An Fab, F(ab')₂ or Fv fragment of themonoclonal antibody of claim
 1. 3. The monoclonal antibody of claim 1conjugated to a label capable of producing a detectable signal.
 4. Themonoclonal antibody of claim 3 in which the label comprises afluorescer.
 5. The monoclonal antibody of claim 1 which comprises an IgGisotype.
 6. The monoclonal antibody of claim 1 which is produced by amurine hybridoma cell line.
 7. The monoclonal antibody of claim 6 inwhich the murine hybridoma comprises a hybridoma having the identifyingcharacteristics of HB8626 as deposited with the A.T.C.C.
 8. An Fab,F(ab')₂ or Fv fragment of the monoclonal antibody of claim
 7. 9. Amonoclonal antibody, the antigen-combining site of which binds to aprotein antigen having a molecular weight of about 140,000 daltons andwhich is a cell-surface determinant of human non-small cell lungcarcinoma.
 10. An Fab, F(ab')₂ or Fv fragment of the monoclonal antibodyof claim
 9. 11. The monoclonal antibody of claim 9 conjugated to a labelcapable of producing a detectable signal.
 12. The monoclonal antibody ofclaim 11 in which the label comprises of fluorescer.
 13. The monoclonalantibody of claim 9 which comprises an IgM isotype.
 14. The monoclonalantibody of claim 9 which is produced by a murine hybridoma cell line.15. The monoclonal antibody of claim 14 in which the murine hybridomacomprises a hybridoma having the identifying characteristics of HB8627as deposited with the A.T.C.C.
 16. An Fab, F(ab')₂ or Fv fragment of themonoclonal antibody of claim
 15. 17. A monoclonal antibody, theantigen-combining site of which binds to a protein antigen having amolecular weight of about 72,000 daltons and which is a cell-surfacedeterminant of human non-small cell lung carcinoma.
 18. An Fab, F(ab')₂or Fv fragment of the monoclonal antibody of claim
 17. 19. Themonoclonal antibody of claim 17 conjugated to a label capable ofproducing a detectable signal.
 20. The monoclonal antibody of claim 19in which the label comprises a fluorescer.
 21. The monoclonal antibodyof claim 17 which comprises an IgG isotype.
 22. The monoclonal antibodyof claim 17 which is produced by a murine hybridoma cell line.
 23. Themonoclonal antibody of claim 22 in whih the murine hybridoma comprises ahybridoma having the identifying characteristics of HB8628 as depositedwith the A.T.C.C.
 24. An Fab, F(ab')₂ or Fv fragment of the monoclonalantibody of claim 23.